Modified dog alpha1-interferon gene, construction method of expression vector and preparation method of protein encoded by modified dog alpha1-interferon gene

An expression vector and interferon technology, applied in the field of construction and protein preparation, can solve problems such as unfavorable industrial production of canine α1-interferon, no technical solution, and inability to fundamentally solve the cost problem of canine α1-interferon , to achieve the effect of improved antiviral activity, low cost and simple method

Inactive Publication Date: 2015-09-09
SOUTH CHINA AGRI UNIV
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Problems solved by technology

[0004] Although some achievements have been made in the use of yeast to express interferon, how to modify the interferon gene to make it biased from yeast for different genes and homologous genes from different species Phenotropic matching to increase the expression of interferon; how to cultivate during the expression process, how to ensure the dissolved oxygen required for protein expression, how to avoid pollution, the amount and time of methan

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  • Modified dog alpha1-interferon gene, construction method of expression vector and preparation method of protein encoded by modified dog alpha1-interferon gene
  • Modified dog alpha1-interferon gene, construction method of expression vector and preparation method of protein encoded by modified dog alpha1-interferon gene
  • Modified dog alpha1-interferon gene, construction method of expression vector and preparation method of protein encoded by modified dog alpha1-interferon gene

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[0050] Example 1

[0051] 1. Design and synthesize a pair of primers according to the nucleotide sequence of canine α1-interferon to synthesize canine α1-interferon gene;

[0052] The present invention refers to the literature (Gurkan C, 2005; Zhao Xiang, 2000) to analyze the codon preference of the Pichia pastoris expression system, and bias the whole gene sequence of canine interferon α1 published in Genbank (A33693) Transformation of addiction, while adding separately upstream and downstream EcoR I and Xba I restriction site (underlined part) and protective base, and in EcoR A base G is added upstream of the I restriction site. The modified gene was synthesized by Shanghai Bioengineering Co., Ltd., and the modified canine α1-interferon gene was inserted into the multiple cloning site of plasmid pUC57. The recombinant plasmid was named pUC57-CaIFN-α1. According to the modified canine interferon α1 nucleotide sequence, a pair of primers are designed and synthesized:

[0053] ...

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Abstract

The invention discloses a modified dog alpha1-interferon gene and a preparation method of dog alpha1-interferon. The modified dog alpha1-interferon gene has the nucleotide sequence as shown in SEQ ID NO.1 and the amino acid sequence coded by the gene is shown in SEQ ID NO.2. The invention concretely relates to matching and modification for the predilection of a dog alpha1-interferon codon to a yeast codon as well as optimal control on a culture method for efficiently producing dog alpha1-interferon, pollution avoiding measures and operation methods of methanol adding amount and time determination, protein expression time control, namely protein harvest time, sterility test, protein content and activity measurement, culture condition expansion and the like. The method disclosed by the invention is simple and easy to realize, relatively low in cost, and capable of realizing stable and efficient expression of the dog alpha1-interferon in pichia pastoris, and the expressed dog alpha1-interferon has a remarkable effect on treating dog viral infectious diseases and can enhance the immune effect of a rabies vaccine.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals. More specifically, it relates to a modified canine α1-interferon gene, the construction of expression vector and the preparation method of its protein. Background technique [0002] Interferon (IFN) is a glycoprotein with broad-spectrum antiviral activity produced by cells under the action of a specific inducer. When it acts on other cells again, it can immediately obtain antiviral, antitumor and other aspects immunity. A large number of in vitro and in vivo tests have shown that canine α1-interferon has protective and inhibitory effects on viral infectious diseases that are a major threat to production. Interferon has broad application prospects due to its wide range of effects and small residues. However, due to the high cost of interferon and the economic value of animals, its clinical application in veterinary medicine is mainly limited to the treatment of pet diseases, and the ...

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Application Information

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IPC IPC(8): C12N15/21C12N15/81C12N1/19C07K14/56
Inventor 郭霄峰杨谦王玉彭娇娇
Owner SOUTH CHINA AGRI UNIV
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